Mitogenic properties of a bispecific single-chain Fv-Ig fusion generated from CD2-specific mAb to distinct epitopes

The combination of anti-CDS mAb 9.6 and 9-1, specific for distinct epitopes , induces proliferation of resting human T cells. The mitogenic activity of this mAb mixture depends upon accessory cells and the 9-1 mAb Fc domain. T o further study the functional properties of these mAb, their variable regi ons were cloned and expressed as monospecific single-chain Fv (scFv) protei ns fused to the human IgG1 Fc domain (scFvIg). A novel bispecific scFvIg wa s constructed by cloning the two monospecific scFv binding sites in tandem, with the 9.6 scFv placed N-terminal to the 9-1 scFvIg, Monospecific scFvIg binding to CD2 was comparable to that of the corresponding parental mAb, w hile the bispecific scFvIg exhibited binding activity similar to that of th e 9-1 scFvIg. The combination of 9.6 scFvIg and 9-1 mAb was mitogenic, wher eas mixtures including the 9-1 scFvIg were non-stimulatory, confirming the unique properties of the 9-1 IgG3 Fc. Without the IgG3 tail, the bispecific 9.6/9-1 scFvIg was directly mitogenic and was a more potent mitogen than t he mAb mixture, but was accessory cell dependent. Unlike the combination of mAb, the bispecific reagent did not directly mobilize calcium in T cells, In comparison to the mAb mixture, bispecific 9.6/9-1 scFvIg-mediated stimul ation of a mixed lymphocyte reaction was significantly more resistant to in hibition of the CD28 co-stimulatory pathway by the inhibitor CTLA-4-Ig. The se results show that expression of the 9.6 and 9-1 binding sites together o n a bispecific scFvIg increased the mitogenic properties of the mAb and alt ered the degree of accessory cell signals required for T cell activation.